1. Field of the Invention
This invention relates to a capillary structure and a method for controlling electroosmotic flow (EOF) in a capillary electrophoresis apparatus.
2. Description of the Related Art
Capillary electrophoresis (CE) is a well-known analytical technique in which the components (ionic species) of an analyte are separated based on their ratio of charge to mobility. In a typical capillary zone electrophoresis (CZE) system (a common implementation of CE), the analyte is introduced along with a carrier electrolyte or buffer into one end of a small-bore capillary and a strong electrical separation potential (an electric field) is applied axially over the length of the capillary. More highly charged and more mobile components of the analyte then tend to move faster through the capillary, so that components are separated into different bands or regions. A detector, which is usually light-based, is then used to sense the passage of each band past a detection region and to determine the speed of migration of the various bands (also known as "peaks") through the capillary. The absolute and relative speeds of migration, as well as spectrographic or other detector information, can then be compared with known patterns to determine in part which chemical or biochemical components are in the analyte.
Ideally, the various components or species in the analyte separate into narrow, well-separated bands: if adjacent bands are too broad, they may overlap and make it impossible even to distinguish them.
Capillary electrophoresis is most often performed in circular capillaries fifty microns in inside diameter, as this size has been found empirically to give favorable performance tradeoffs. This size is large enough to allow sensitive detection, yet not so large as to produce excessive radial heating and viscosity gradients which broaden bands and destroy resolution.
At this fifty-micron capillary diameter, electroosmotic flow (EOF) produces a well-developed plug-shaped flow. At the edge of the capillary, flow is laminar and in sheer, but the thickness of this double-layer region is on the order of microns with commonly used buffers and pH's. The flow velocity profile therefore rises rapidly at the outer microns of the flow plug, and then transitions to a fairly flat region across the wide center of the flow profile. Thus, most of the electrolyte flows at the same velocity, and undesirable laminar mixing, which would broaden the peaks, is minimized. The penalty for this operating regime is, however, substantial EOF, which is discussed in greater detail below.
Capillary tubes used in CE are normally made of fused silica. Above about pH 4, negative ions in the carrier electrolyte attach to silanol groups of the fused silica and leave a concentration of positively charged ions near the radially inner side of the double layer. The resulting layer of negative ions attached to the silica surface and the layer of positive ions extending away from the surface form the well-known "double layer" structure near the inner capillary wall. Under the influence of the large electric field applied axially along the capillary, this positive ionic charge concentration results in considerable undesirable pumping forward towards the cathode, that is, toward the end of the capillary that is at the "negative" region of the applied electric field. Electroosmotic flow (EOF) is the bulk flow in the separation capillary that is caused by this pumping, that is, that results from the ions within the double layer being propelled by the axial separation potential. This potential also carries along the neutrally charged inner region of buffer in the capillary.
A significant problem associated with EOF is that it may cause the analyte to move through the capillary so fast that the components of the analyte do not have time to separate clearly enough to distinguish and identify them. Although some EOF may be desirable so as to allow species of both positive and negative ionic charge to reach the detector, there has consequently been a long-standing and frequently articulated need to reduce or control EOF.
One way to offset the effect of EOF is simply to make the capillary longer so that the components of the analyte will have farther to travel and therefore more time in which to be separated, provided, however, that the potential along the capillary is increased proportionately. On the other hand, one typically wishes to make the capillary shorter in order to permit the use of more affordable power supplies: the longer the capillary is, the stronger the separation potential must be in order to achieve the same potential gradient or field.
Other conventional attempts to control EOF have been only partially successful, or successful only under a narrow range of conditions. One known method involves limiting separations to only those with low buffer pH, which limits the usefulness of the CE device. Another method involves greatly increasing the buffer concentration; however, this increases Joule heating, which worsens CE resolution. Yet another method involves increasing buffer viscosity, but this may alter the selectivity of the device. Still another method involves chemically bonding some compound to the capillary wall to terminate the silanol sites, but such coatings do not remain stable.
According to another category of methods for controlling EOF, at least one radial electric field is applied to the capillary by coaxial conductors or resistors. Such fields include tracking fields and sheath fields of constant potential. Experience has shown, however, that neither of these configurations is effective over a wide pH range.
The superposition of laminar flow upon EOF is discussed in the article "Electrokinetic Dispersion in Capillary Electrophoresis," Ravindra Datta and Veerabhadra Kotamarthi, AIChE Journal, Vol. 36, No. 6, June 1990. In this article it is shown that radial viscosity changes from Joule heating produce a slightly convex "plug" flow profile, which can be flattened by application of a mild back pressure. By setting the back pressure properly, lower dispersion was realized and plate height minimized, that is, the separation bands were narrowed.
It is conventionally believed that strong back pressure reduces the sharpness of zone boundaries, that is, that it causes band-broadening. The assumed reason is that the back pressure causes a counterflow or eddy-currents along the capillary: electroosmotic forces act primarily at the outer edges of the plug and propel that region towards the detector whereas hydrostatic forces act across the entire cross section and tend to develop a parabolic back flow, which is greatest at the center of the capillary. The capillary therefore continually tends to mix its contents, swirling the outer edges forward and the center region backward.
In the context of isotachophoresis, Everarts, Verheggen, and Van De Veene have shown by ,experiment that small back flows (on the order of 10%) improve the plug flow profile and sharpen the zones. See "Isotachophoretic Experiments with a Counter Flow of Electrolyte," 123 Journal of Chromatography, pp. 139-148, 1976. They stated, however, that they found that if a 100% back flow of electrolyte is applied (such that the hydrodynamic back flow of electrolyte is in equilibrium with the electrophoretic flow), the sharpness of the zone boundaries was lost. They also reported that even at 50-60% back flow of electrolyte, many zones became mixed. Of note is that these experiments were carried out using a separation capillary that had an inner diameter of 500 microns, which is ten times larger than the capillaries normally used in capillary electrophoresis.
The pressure required to induce a 10% back flow in a conventional capillary is on the order of 0.01 atmosphere, or a centimeter head of water. Although these known hydrostatic methods strive to increase resolution in different ways and under different operating conditions, they fail to deal with the problem of controlling or eliminating EOF.
Small-bore capillaries for CE are known to have the advantage that higher electric fields may be applied with less problem of developing radial temperature gradients. On the other hand, these small-bore capillaries normally suffer from greatly reduced sensitivity, since their capacity is lower and drops as the square of the capillary diameter. For example, a fifteen-micron capillary delivers less than a tenth as much sample to a detector as a fifty-micron capillary. Furthermore, detection using the smaller capillary is much more difficult, since the detection path length for UV absorbance detectors is then less than one-third as long, and the detection cell is so narrow that it is hard to focus the detection light into it without stray light worsening the desired linearity.
Another limitation of small-bore capillaries is that their few-micron double-layer region of laminar flow does not scale with size, but rather becomes a significant percentage of the total flow, thus contributing to laminar mixing and loss of resolution. For example, a 21/2-micron double layer region represents less than 20% of the cross-sectional area of a fifty-micron capillary, but 75% of the area of a ten-micron capillary, thus causing very serious laminar flow and peak broadening.